PROJECT SUMMARY Sarcomas are an immunogenic tumor type replete with tumor antigen experienced CD8 tumor infiltrating lymphocytes (TILs). Surprisingly, checkpoint therapy that targets programmed cell death protein 1 (a-PD-1) to reinvigorate CD8 TILs is largely ineffective in sarcomas and has not gained FDA approval. CD8 TILs experience stress, but the stress response has not been widely studied in CD8 TILs of cancer patients. Under acute stress, endoplasmic reticulum (ER) stress sensor protein kinase R (PKR)-like ER kinase (PERK) protects cells. Under chronic stress, PERK activates a pro-apoptotic response that induces cell death. We previously demonstrated that PERK is detrimental to CD8 T cells in tumors. We now reveal robust preliminary data that indicate that the chronic PERK axis governed by activating transcription factor 4 (ATF4) and ER oxidoreductase 1 (ERO1a) shapes CD8 TIL fate in mouse and human sarcomas and impairs response to a-PD-1 therapy. Our data are intriguing given the role of PERK to protect cells under acute stress through attenuation of translation. Indeed, we found that the tumor microenvironment is a form of acute stress that inhibits translation in CD8 T cells through PERK. However, in vivo in the presence of tumor antigen, the chronic arm of the PERK response appears to dictate PD-1+ CD8 TIL fate. This proposal will formally test that chronic targets ATF4 and ERO1a drive activation and metabolic exhaustion in CD8 TILs that limit the efficacy of a-PD-1 therapy, while the acute PERK response protects CD8 TILs under sarcoma microenvironment stress. To accomplish our aims we have developed unique genetic mouse models to analyze the T cell-specific contributions of the chronic and acute phases of the stress response to shape efficacy of a-PD-1 therapy in sarcomas. In Aim 1, we will use LckcreRosa26-ATF4loxtg mice with T cell-specific overexpression of human ATF4 and CD8 TILs from sarcoma patients to determine the contribution of ATF4 to drive activation and exhaustion in CD8 TILs and shape response to a-PD-1 therapy in sarcomas. The results are expected to reframe and advance our understanding of T cell exhaustion in sarcomas. In Aim 2 we have created unique ERO1a-/- mice to formally define how ERO1a affects CD8 TIL metabolic exhaustion and response to a-PD-1 therapy and we will use CD8 TILs from sarcoma patients to study the contribution of ERO1a to human TIL exhaustion. The results are expected to produce a robust molecular target that holds fantastic potential to improve the efficacy of a-PD-1 therapy in cancer patients. In Aim 3 we will use our LckcrePERKf/f mice and Eif2aS51A mutant mice to elucidate requirements of the acute stress response in CD8 TILs. The results are expected to shape the direction of drug development surrounding the ER stress response in cancer immunotherapy. Successful completion of this proposal will identify radical new chronic ER stress targets that undermine the widespread success of immunotherapy in sarcoma patients and establish a new paradigm that informs drug development for all solid tumor cancer patients.